Tight regulation of cellular and plasma cholesterol is crucial to proper cellular functioning, as excess free cholesterol is toxic to cells and is associated with atherosclerosis and heart disease. Cellular cholesterol homeostasis is regulated by enzymatically formed oxygenated cholesterol derivatives called oxysterols. Oxysterols can suppress expression of genes responsible for de novo cholesterol synthesis and lipoprotein cholesterol uptake, and serve as endogenous ligands for nuclear receptors, which activate cholesterol catabolic and efflux pathways. While the importance of oxysterols in the acute regulation of cholesterol homeostasis is known, the precise molecular mechanisms through which oxysterols exert their effects remain to be elucidated. My central hypothesis is that oxysterols exert their cholesterol-regulatory effects through non- enantioselective membrane-disordering effects. To test this hypothesis, I will (1) synthesize both the natural (nat) and enantiomeric (ent) forms of LY295427-a known antagonist of 25-HC, (2) examine the enantioselectivity of nat- and ent-LY295427-membrane interactions and the effect of LY295427 on the membrane-disordering properties of 25-HC, and (3) determine the enantioselectivity of LY295427 inhibition of 25-HC-mediated sterol homeostatic responses in cultured cells. The long-term objectives of this project are to elucidate the molecular mechanisms underlying the maintenance of cellular cholesterol balance, and to provide insight into how alteration in membrane structure can be used to relay regulatory signals. The proposed studies may help to identify pharmacological targets for manipulation of the cellular handling of cholesterol and treatment of atherosclerosis. PUBLIC HEALTH RELEVANCE: High levels of plasma lipoprotein cholesterol (e.g., in the form low-density lipoproteins (LDL), are associated with atherosclerosis and heart disease. Understanding the mechanisms through which cellular and whole-body cholesterol levels are regulated will provide new approaches to developing cholesterol-lowering treatments, and may reduce cardiovascular disease.